Baseline difference between HPLC and UPLC

<p>I recently was involved in developing an assay for which we needed equivalent HPLC and UPLC methods. I started with an HPLC method and then used the Waters Acquity calculator to achieve equivalent UPLC conditions. Both HPLC and UPLC give similar sensitivity and separation, but the baseline profiles are significantly different (HPLC goes up but UPLC goes down during the gradient). This has been confirmed on several instruments. Does anyone know what could cause the baseline to be so different? Figures attached.</p><p></p><p>MPA: 0.05% phosphoric acid</p><p>MPB: ACN</p><p>Column temp: 30 C</p><p>Dectection: 260 nm</p><p></p><p>HPLC: Waters 2695</p><p>Column: XSelect HSS T3</p><p>Flow: 1.5 mL/min</p><p></p><p>UPLC: Waters H-Class</p><p>Column: Acquity HSS T3</p><p>Flow: 0.5 mL/min</p>


  • Hi

    Which detectors are you using on the systems? Is this a gradient based method? If it is, what is the method and the total injection to injection cycle time?

    Ken Blakeslee

  • This is a gradient method (ends at about 80% MPB).

    The HPLC uses a 2998 PDA detector. HPLC run time is 28 min with 6 min re-equilibration. Injection-to-injection ends up being about 35 min.

    The UPLC uses and Acquity PDA detector. UPLC runtime is 11.5 min with 2.5 min re-equilibration. Injection-to-injection ends up being about 15 min.

  • I believe the difference in the baselines is caused by flow cell geometry.

    The HPLC detector is designed to direct the light beam through the center of the flow cell, avoiding the flow cell walls.

    The UPLC flow cell is designed differently, relying on total internal reflection of light through the flowcell - a light guided flowcell. As the flow cell ages, the properties of the Teflon AF (changed typo from AG to AF) coating can change, resulting in baselines that have a swoop or smile shape, like the UPLC baseline you've provided.

    You can compensate for this in Empower by using the Median Baseline Filter or Reference Compensation.

    If you have a copy of the ACQUITY H-Class for practitioners training manual, this is covered in the Detection Mechanisms section.


  • Thank you for the helpful response! Do you know if that manual is available online?

  • I don't think you'll find that reference online, but if Waters provided operator training after they installed the systems, they would probably have supplied a printed copy.


  • Generating a Median Baseline Filter channel is done by selecting this setting in the Acquity Instrument Method for the detector in the 2D tab I believe. You can designate your desired wavelength with conventional absorbance and with MBF for comparison.

    The two attached documents describe the light guided flow cell and include care and use, plus maintenence guidelines.

    Ken Blakeslee